Boletus edulis extract as well as preparation method and application thereof

Abstract

The invention discloses a boletus edulis extract as well as a preparation method and application thereof, and provides application of the boletus edulis extract in preparation of medicines, health-care products or functional foods for preventing and / or treating senile intestinal dysfunction caused by colonic atrophy and degeneration. The preparation method of the boletus edulis extract comprises the following steps: crushing boletus edulis, extracting with water or an alcohol solvent, filtering to obtain an extracting solution, concentrating the extracting solution, and drying to obtain the boletus edulis extract. The boletus edulis extract provided by the invention can up-regulate the expression of tight junction protein ZO-1 and mucoprotein MUC-2 in the colon of an aging body and promote the growth of colon length and crypt depth, thereby delaying the degeneration of colon atrophy and enhancing the digestive absorption and immune function of intestinal tracts.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine and functional food, in particular to a Boletus edulis extract, a preparation method and application thereof, and more particularly to the Boletus edulis extract, a preparation method and application thereof in the preparation of drugs, health products or functional food for preventing and / or treating senile intestinal dysfunction caused by colon atrophy. BACKGROUND

[0002] With the intensification of global population aging, age-related decline in digestive system function has become an important problem affecting the quality of life of the elderly. In terms of colon, aging is usually accompanied by atrophic changes in colon tissue, manifested as shortening of colon villi and shallowing of crypt depth, leading to decreased absorption efficiency of nutrients. More importantly, the intestinal barrier function is impaired, characterized by down-regulation of expression of tight junction proteins (such as ZO-1) and mucins (such as MUC2). This can lead to increased intestinal permeability ("leaky gut"), triggering chronic low-grade inflammation, and weakening the intestinal immune defense, which is closely related to the occurrence and development of many senile diseases.

[0003] Currently, there are limited interventions for senile intestinal function decline, mainly through the supplementation of probiotics, prebiotics or dietary fiber. However, the effects of these methods vary from person to person, and have limited effect on fundamentally improving intestinal structure and barrier function. Therefore, it is of great clinical significance and market value to develop a new type of natural product that can effectively delay colon degeneration and strengthen the physical and immune barrier of the intestine.

[0004] Boletus edulis is a precious edible fungus rich in polysaccharides, terpenes, phenols and other bioactive components. Existing researches mainly focus on its antioxidant, immune regulation or anti-tumor activity, but there is no report on using its specific extract to specifically improve the morphological and functional decline of the colon related to aging. SUMMARY

[0005] The purpose of the present application is to provide a Boletus edulis extract that enhances the intestinal immunity of the aging body, a preparation method thereof and its application in the preparation of drugs, health products or functional food for preventing and / or treating senile intestinal dysfunction caused by colon atrophy.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: The present application provides the application of a Boletus edulis extract in the preparation of drugs, health products or functional food for preventing and / or treating senile intestinal dysfunction caused by colon atrophy, wherein the preparation method of the Boletus edulis extract is as follows: Boletus edulis is pulverized, extracted with water or an alcoholic solvent, filtered to obtain an extract, and the extract is concentrated and dried to obtain the Boletus edulis extract.

[0007] Preferably, the drug, health product, or functional food delays colonic atrophy and degeneration, and enhances intestinal digestion, absorption, and immune function.

[0008] Preferably, the drug, health product, or functional food promotes the growth of colon length and crypt depth.

[0009] Preferably, the drug, health product, or functional food upregulates the expression of tight junction protein ZO-1 and mucin MUC-2 in the colon of aging organisms.

[0010] Preferably, the water or alcohol solvent extraction is performed by adding 10-30 L of water or alcohol solvent per kilogram of Boletus edulis, wherein the alcohol solvent is a 70%-80% ethanol-water solvent.

[0011] Delicious porcini mushrooms ( Boletus edulis Boletus edulis, commonly known as white boletus or bigfoot mushroom, is one of the safest and top-grade edible fungi recognized globally, and is also one of the very few wild mushrooms that can be eaten raw (but it is still recommended to cook it).

[0012] Preferably, the porcini mushrooms of the present invention undergo freeze-drying treatment before being pulverized.

[0013] The porcini mushrooms are pulverized to 100-200 mesh.

[0014] Preferably, the extraction method may be immersion extraction, hot reflux extraction, ultrasonic extraction, or any combination of two thereof.

[0015] Preferably, the hot reflux extraction time is 1-5 hours and the number of extractions is 1-4 times; more preferably, the hot reflux extraction time is 2-3 hours and the number of extractions is 2-3 times.

[0016] Preferably, the preparation method of the delicious Boletus extract further includes the following purification steps: dissolving the alcohol extract in water, extracting with ethyl acetate, collecting the ethyl acetate extract, concentrating, and drying.

[0017] Preferably, in the ethyl acetate extraction, the volume ratio of the aqueous phase to ethyl acetate is 1:1 to 5, and the extraction is performed 2 to 4 times.

[0018] Preferably, the drug further comprises one or more pharmaceutically acceptable carriers or excipients.

[0019] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: sustained-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, and lubricants. The combination of at least two is, for example, a combination of binders and excipients, a combination of binders and flavoring agents, a combination of binders and fillers, etc. Other combinations are also possible and will not be elaborated here.

[0020] Preferably, the health product or functional food further comprises one or more food-acceptable carriers or excipients.

[0021] Preferably, the dosage form of the drug or health product is tablet, injection, capsule, granule, pill, decoction, syrup or mixture.

[0022] The beneficial effects of this invention are as follows: This invention is the first to discover that *Boletus edulis* extract can specifically improve the morphological and functional decline of the colon caused by aging, with a clear mechanism of action. Experimental results demonstrate that *Boletus edulis* extract can significantly promote the increase of colon length and crypt depth, reversing colonic atrophy from an anatomical perspective and laying the foundation for improved digestive and absorptive functions. *Boletus edulis* extract can efficiently upregulate the expression of tight junction protein ZO-1, strengthening the connections between intestinal epithelial cells, reducing intestinal permeability, thereby reducing endotoxin translocation and systemic inflammation. *Boletus edulis* extract can promote the high expression of gel-forming mucin MUC2, enhancing the thickness and integrity of the intestinal mucus layer, playing a role in lubricating the intestine, isolating pathogens, and strengthening the first line of immune defense. *Boletus edulis* is an edible fungus with high safety and is easily accepted, making it suitable for development into health foods or pharmaceutical excipients for long-term use. This provides a novel and effective natural solution for the prevention and treatment of age-related intestinal dysfunction caused by colonic atrophy and degeneration. Attached Figure Description

[0023] Figure 1 Results of HE staining and PASS staining of colon tissue; Figure 2 The results show the expression levels of intestinal mucin ZO-1 and MUC2 proteins. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0025] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0026] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0027] The following are some of the material sources used in the examples: Delicious porcini mushrooms ( Boletus edulis Fresh produce, purchased from Yunnan Lijiang Zhongyuan Green Food Co., Ltd.; 95% ethanol, purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd., purity ≥99.5%. Petroleum ether, purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd., with a purity of ≥99.5%; Ethyl acetate, purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd., with a purity of ≥99.5%; Dexamethasone, purchased from Beijing Solarbio Biotechnology Co., Ltd., with a purity of ≥99.5%; Methotrexate, purchased from Beijing Solarbio Biotechnology Co., Ltd., with a purity of ≥99.5%; C57BL / 6 mice, SPF grade, were purchased from the Animal Experiment Management Center of Yunnan University.

[0028] Example 1 Fresh Boletus edulis was washed with deionized water, sliced, freeze-dried to constant weight, and then pulverized to 200 mesh to obtain Boletus edulis powder. An appropriate amount of powder was taken and added to 75% ethanol solution at a material-to-liquid ratio of 1:20 (g / mL). The mixture was extracted three times at 75℃ under reflux (2 h each time). The extracts were combined and concentrated under reduced pressure to obtain Boletus edulis extract.

[0029] Example 2 Fresh Boletus edulis was washed with deionized water, sliced, freeze-dried to constant weight, and then pulverized to 200 mesh to obtain Boletus edulis powder. An appropriate amount of powder was taken and water was added at a material-to-liquid ratio of 1:20 (g / mL). The mixture was extracted three times by hot reflux (2 h each time). The extracts were combined and concentrated under reduced pressure to obtain Boletus edulis extract.

[0030] Example 3 Fresh Boletus edulis was washed with deionized water, sliced, freeze-dried to constant weight, and then pulverized to 200 mesh to obtain Boletus edulis powder. An appropriate amount of powder was taken and added to 70% ethanol solution at a material-to-liquid ratio of 1:10 (g / mL). The mixture was extracted twice by hot reflux (3 h each time). The extracts were combined and concentrated under reduced pressure to obtain Boletus edulis extract.

[0031] Example 4 Fresh Boletus edulis was washed with deionized water, sliced, freeze-dried to constant weight, and then pulverized to 200 mesh to obtain Boletus edulis powder. An appropriate amount of powder was taken and added to 80% ethanol solution at a material-to-liquid ratio of 1:30 (g / mL). The mixture was extracted twice by hot reflux (2 h each time). The extracts were combined and concentrated under reduced pressure to obtain Boletus edulis extract.

[0032] Example 5 The Boletus edulis extract from Example 1 was dissolved in water and extracted with 3 times its volume of ethyl acetate. The extraction was repeated 3 times. The extracts were combined and concentrated under reduced pressure using a rotary evaporator and dried to obtain the Boletus edulis ethyl acetate extract.

[0033] Example 6 The Boletus edulis extract from Example 1 was dissolved in water and extracted with 5 times its volume of ethyl acetate. The extraction was repeated twice. The extracts were combined and concentrated under reduced pressure using a rotary evaporator and dried to obtain the Boletus edulis ethyl acetate extract.

[0034] Example 7 The Boletus edulis extract from Example 1 was dissolved in water and extracted with 1 volume of ethyl acetate. The extraction was repeated 4 times. The extracts were combined and concentrated under reduced pressure using a rotary evaporator and dried to obtain the Boletus edulis ethyl acetate extract.

[0035] Experiment Example 1: Effects of Aging on Gut Immunity 1. Test sample Examples 1-7 show the preparation of extract samples.

[0036] 2. Experimental animals C57BL / 6 mice, 6-8 weeks old, SPF grade.

[0037] 3. Test methods 3.1 Modeling After one week of acclimatization feeding, 10 C57BL / 6 mice were randomly selected as a blank control group and injected with 0.5 mg / kg·bw physiological saline once a day. The remaining C57BL / 6 mice were injected intraperitoneally with 25 mg / kg·bw dexamethasone injection once a day to establish an aging intestinal mouse model. From day 1 to day 21, the dietary and weight changes of C57BL / 6 mice were measured every two days, and the changes in the mice's tensile strength were monitored.

[0038] 3.2 Intervention on test samples After modeling, the experimental group was randomly divided into 9 groups, with 10 C57BL / 6 mice in each group. Groups 1-7 were administered the extracts of Examples 1-7 of this invention by gavage (dose of 60 mg / kg bw, dissolved in water to a concentration of 12 mg / ml before gavage), once daily. Group 8 served as the model control group, receiving an equal volume of physiological saline by gavage once daily. Group 9 served as the positive control group, receiving an equal volume of methotrexate treatment by gavage once daily. The test samples were weighed 21 days after intervention. Calculate colon length, detect colon length and crypt depth.

[0039] 3.3 Colonic Intestinal Examination: Colonic HE Staining (Hematoxylin-eosin Staining) Experiment Colon tissues from the blank control group, model group, Example 1 group, and Example 5 group were sectioned in paraffin. The paraffin sections were rinsed with distilled water after being treated with xylene I and II for 10-20 minutes each, followed by anhydrous ethanol I and II for 10 minutes each, and then 95%, 90%, 80%, and 70% ethanol for 5 minutes each. The sections were then fixed with 4% paraformaldehyde for 24 hours. They were then immersed in hematoxylin stain for 3 minutes, rinsed with tap water for 3 minutes, separated with 1% hydrochloric acid ethanol for 10 seconds, rinsed with running water for 1 minute, immersed in eosin stain for 2 minutes, and then gently rinsed with tap water for 1 minute. Finally, a gradient dehydration process was performed: 80%, 95%, and anhydrous ethanol for 2-5 minutes each, followed by clearing with xylene twice for 5 minutes each time, mounting with neutral resin, and examining under a microscope.

[0040] 3.4 Colonic Intestinal Detection: PAS Staining (Periodic Acid-Schiff Staining) Experiment for Colonic Glycoconjugates Dewax paraffin sections to water (same procedure as HE staining). Oxidize sections with 1% periodic acid solution for 5-10 minutes, then rinse with running water for 5 minutes. Immerse in Schiff's reagent for 15 minutes, then rinse with running water for 7 minutes (to remove excess reagent). Counterstain with Mayer's hematoxylin for 1 minute, rinse with running water to regain blue color, dehydrate in a gradient (same as HE), clear with xylene, mount with neutral resin, and examine under a microscope.

[0041] 3.5 Detection of expression levels of intestinal mucin ZO-1 and MUC2 proteins.

[0042] (1) Extraction of colonic protein tissue from blank control group, model group, Example 1 group and Example 5 group of mice: Weigh about 20 mg of mouse colonic tissue sample and place it in a 2 ml round bottom EP tube, add 2 steel balls, add 1 mL of 1% PMSF cell lysis buffer to each tube, and break it in a tissue homogenizer for 30 s / time; after complete disruption, let it stand on ice for 30 min, centrifuge at 4℃ and 12000 rpm for 15 minutes; transfer the supernatant to a new 1.5 mL centrifuge tube, repeat the centrifugation operation until the supernatant is clear and transparent, and store at -80℃ for later use.

[0043] (2) Quantitative sample preparation: The concentration of protein in the sample was detected using the BCA kit. First, a standard curve was prepared. In a 96-well dilution plate, the 1 mg / ml standard protein sample was diluted sequentially (0.5, 0.25, 0.125, 0.0625, 0.03125). Filtered PBS was added to the blank wells and marked as 0. 2 µL of the test protein was added to 38 µL of filtered PBS and diluted 40 times, then mixed using a multi-channel pipette. 20 µL of the mixed standard protein and test protein were transferred to the microplate using a multi-channel pipette, with three replicates for each sample. 200 µL of BCA working solution (solution A:solution B = 50:1) was added to each well, and the plate was incubated at 37℃ for 30 min. The absorbance of the microplate was measured at 590 nm and 630 nm, and the standard curve was calculated. The absorbance of the test sample protein was substituted into the standard curve to calculate the sample protein concentration. After calculation, a reducing sample carrier solution (3-5 µL of 5× reducing sample carrier solution was added to 30 μg of total protein) was added to the sample protein. Then, the sample was boiled in a 95℃ sample cooker for 5-10 min to denature the protein. The sample was then allowed to cool naturally at room temperature. Membrane proteins were boiled at 37℃ for 30 min.

[0044] (3) Sample loading: Prepare 1× electrophoresis buffer, 8% separating gel, and the prepared protein sample according to the molecular weight of the target protein. Fill the electrophoresis tank with the gel plate in place with the electrophoresis buffer, slowly pull out the gel plate comb from the electrophoresis tank, clean the residual gel particles in the lanes, and prepare for sample loading. Use a 20 µL pipette to draw up the protein sample and slowly and evenly add it to the sample well (ensuring that the sample protein is placed in the middle of the well as much as possible). Fill the remaining wells with 5× reducing sample loading solution.

[0045] (4) Electrophoresis: Set the electrophoresis program. a. First program: constant voltage 50 V, time 30 minutes; b. Second program: constant voltage 120 V, time 90 minutes. Observe the pre-stained marker on the sample according to the required protein size to terminate electrophoresis.

[0046] (5) Transfer: After marking the PVDF membrane and activating it in methanol for 30 seconds, transfer it to cold, pre-prepared 1x transfer buffer to wash off the methanol. After the electrophoresis program, remove the upper layer of stacking gel and prepare a sandwich structure in the following order: sponge, 4 layers of filter paper, PVDF membrane, separating gel, 4 layers of filter paper, sponge (avoid air bubbles between the separating gel and the PVDF membrane). Use clamps to clamp the sandwich structure and ensure that the positive and negative electrodes of the clamps are placed in the transfer tank. At the same time, pay attention to the positive and negative electrode orientation on the electrode caps. Start the transfer program: constant current 200 mA, time 60 minutes; keep the transfer tank in an ice-water bath.

[0047] (6) Blocking: After the transfer is completed, the PVDF membrane is removed with tweezers, the surface transfer solution is washed off in 1×TBST, and it is placed in a 5% skim milk powder blocking buffer prepared with 1×TBST. It is then shaken slowly at room temperature for at least 1 hour to avoid the appearance of specific bands later.

[0048] (7) Primary antibody incubation: After blocking, the blocking solution was recovered, and the residual skim milk powder on the PVDF membrane was washed off with 1×TBST. After rinsing three times, the membrane was washed by shaking quickly on a shaker for 5-10 minutes. This process was repeated three times. After washing, the PVDF membrane was placed in an incubation box, ensuring that the primary antibody was completely submerged in the PVDF membrane. The membrane was then incubated overnight on a shaker at 4°C.

[0049] (8) Secondary antibody incubation: recover the primary antibody, repeat the membrane washing operation to wash away the residual primary antibody on the membrane; then incubate with the secondary antibody corresponding to the primary antibody at room temperature for 1 hour; repeat the membrane washing operation to wash away the residual secondary antibody on the membrane.

[0050] (9) Exposure: Mix equal volumes of sensitizer and stabilizer solutions A and B in centrifuge tubes in the dark (prepare 1 mL of exposure solution for each membrane). Place 1 mL of the mixed reagent on the exposure apparatus to ensure that the PVDF membrane is fully in contact with the exposure solution and that the membrane surface is uniformly exposed to the solution. The exposure machine automatically exposes the membrane in the dark chamber, observes and saves the images, and uses ImageJ analysis software to perform quantitative analysis on the images.

[0051] 4. Experimental Results Table 1 shows the statistical table of colonic health in C57BL / 6 mice. The results indicate that the body weight of C57BL / 6 mice in Examples 1-7 of this invention was significantly increased compared to the model control group (p < 0.05). At the same dosage, the body weight recovery in Example 5 group was the best, indicating that the ethyl acetate extract of Boletus edulis was more effective in restoring body weight. Regarding colon length, the colon length and colonic crypt depth of Examples 1-7 were longer than those of the model group, indicating that the Boletus edulis extract could delay colonic atrophy in aging mice and prolong the absorption of nutrients by the colon. Among them, Example 5 showed the best effect, indicating that its ethyl acetate extraction could enrich the active substances that improve intestinal dysfunction and remove most of the inactive substances.

[0052] Table 1 Colonic health status of C57BL / 6 mice

[0053] Note: In the table, the superscript # indicates that p < 0.05 compared with the blank control; the superscript * indicates that p < 0.05 compared with the model control; the superscripts a, b, and c indicate that p < 0.05 between different cases corresponding to different letters.

[0054] Figure 1 HE staining and PASS staining of colon tissue. Figure 2The results of detecting the expression levels of the hallmark intestinal mucoproteins ZO-1 (tight junction protein) and MUC2 (gel protein) show that both Example 1 and Example 5 groups can significantly repair the aging condition in the aging colon model and promote intestinal integrity. PASS staining also indicates that both Example 1 and Example 5 groups promote the expression of glycomucoproteins in the colon, improving intestinal lubrication. The images show that Example 5 group is superior to Example 1 group. Comparison between the model control group and the blank control group shows a significant difference in ZO-1 and MUC2 protein expression (p < 0.05). Compared with the model group, Example 1 and Example 5 groups significantly increased the expression levels of ZO-1 and MUC2 proteins. Furthermore, Example 5 group showed a more significant increase than Example 1 group. The extract of this invention can significantly increase the expression levels of ZO-1 and MUC2 proteins in the colon, increase the content of glycomucoproteins in the intestine, reduce the risk of intestinal diseases in aging mice, and maintain intestinal health, demonstrating significant advantages.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a Boletus edulis extract in the preparation of drugs, health products or functional foods for the prevention and / or treatment of senile intestinal dysfunction caused by colonic atrophy and degeneration, wherein the Boletus edulis extract is prepared by pulverizing Boletus edulis, extracting with water or alcohol solvent, filtering to obtain an extract, concentrating the extract and drying to obtain the Boletus edulis extract.

2. The application according to claim 1, characterized in that, The aforementioned drugs, health products, or functional foods can delay colonic atrophy and degeneration, and enhance intestinal digestion, absorption, and immune function.

3. The application according to claim 1, characterized in that, The drugs, health products, or functional foods mentioned promote the growth of colon length and crypt depth.

4. The application according to claim 1, characterized in that, The drugs, health products, or functional foods mentioned above upregulate the expression of tight junction protein ZO-1 and mucin MUC-2 in the colon of aging organisms.

5. The application according to claim 1, characterized in that, The water or alcohol solvent extraction is performed by adding 10-30L of water or alcohol solvent per kilogram of Boletus edulis, wherein the alcohol solvent is 70%-80% ethanol-water solvent.

6. The application according to claim 1, characterized in that, The extraction method may be immersion extraction, hot reflux extraction, ultrasonic extraction, or any combination of two thereof.

7. The application according to claim 1, characterized in that, The preparation method of the delicious Boletus extract also includes the following purification steps: dissolving the alcohol extract in water, extracting with ethyl acetate, collecting the ethyl acetate extract, concentrating, and drying.

8. The application according to claim 7, characterized in that, In the ethyl acetate extraction, the volume ratio of the aqueous phase to ethyl acetate is 1:1 to 5, and the extraction is performed 2 to 4 times.

9. The application according to any one of claims 1-8, characterized in that, The drug also includes one or more pharmaceutically acceptable carriers or excipients.

10. The application according to any one of claims 1-8, characterized in that, The health products or functional foods also contain one or more food-acceptable carriers or excipients.

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